Enhanced expression in plants

ABSTRACT

This invention provides HSP70 introns that when present in a non-translated leader of a chimeric gene enhance expression in plants.

This is a continuation of application Ser. No. 08/333,665 filed Nov. 3,1994 which issued as U.S. Pat. No. 5,593,874 on Jan. 14, 1987; which isa continuation of application Ser. No. 08/181,364 filed Jan. 13, 1994which issued as U.S. Pat. No. 5,424,412 on Jun. 13, 1995; which is acontinuation of application Ser. No. 07/855,857 filed on Mar. 19, 1992,now abandoned.

This invention relates to recombinant expression systems, particularlyto plant expression systems for expressing greater quantities ofproteins in plants.

BACKGROUND OF THE INVENTION

Recombinant genes for producing proteins in plants comprise in sequencea promoter which functions in plants, a structural gene encoding thetarget protein, and a non-translated region that functions in plants tocause the addition of polyadenylated nucleotides to the RNA sequence.Much scientific effort has been directed to improve these recombinantplant genes to express larger amounts of the target protein.

One advantage of higher levels of expression is that fewer numbers oftransgenic plants need to be produced and screened to recover plantsproducing sufficient quantities of the target protein to beagronomically significant. High level expression leads to plantsexhibiting commercially important phenotypical properties.

Improved recombinant plant genes have been found by use of more potentpromoters, such as promoters from plant viruses. Further improvement inexpression has been obtained in gene constructs by placing enhancersequences 5' the promoter. Still further improvement has been achieved,especially in monocot plants by gene constructs having introns in thenon-translated leader positioned between the promoter and the structuralgene coding sequence. For example, Callis et al. (1987) Genes andDevelopment, Vol. 1, pp. 1183-1200, reported that the presence ofalcohol dehydrogenase-1 (Adh-1) introns or Bronze-1 introns resulted inhigher levels of expression. Dietrich et al. (1987) J. Cell Biol., 105,p. 67, reported that the 5' untranslated leader length was important forgene expression in protoplast. Mascarenkas et al. (1990) Plant Mol.Biol., Vol. 15, pp. 913-920, reported a 12-fold and 20-fold enhancementof CAT expression by use of the Adh-1 intron. Vasil et al. (1989) PlantPhysiol., 91, pp. 1575-1579, reported that the Shrunken-1 (Sh-1) introngave about 10 times higher expression than constructs containing theAdh-1 intron. Silva et al. (1987) J. Cell Biol., 105, p. 245, reported astudy of the effect of the untranslated region of the 18 Kd heat shockprotein (HSP18) gene on expression of CAT. Semrau et al. (1989) J. CellBiol., 109, p. 39A, and Mettler et al., N.A.T.O. Advanced StudiesInstitute on Molecular Biology, Elmer, Bavaria (May 1990) reported thatthe 140 bp intron of the 82 Kd heat shock protein (HSP82) enhancedexpression in maize protoplasts.

The search for even more improved recombinant plant genes continues forthe reasons discussed above.

SUMMARY OF THE INVENTION

This invention is for an improved method for the expression of achimeric plant gene in plants, particularly to achieve higher expressionin monocot plants. The improvement of the invention comprises expressinga chimeric plant gene with an intron derived from the 70 Kd maize heatshock protein (HSP70) selected from the group consisting essentially ofSEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in the non-translated leaderpositioned 3' from the gene promoter and 5' from the structural DNAsequencing encoding a protein.

One embodiment of the invention is a recombinant, double stranded DNAmolecule comprising in sequence:

(a) a promoter that functions in plant cells to cause the production ofan RNA sequence;

(b) a non-translated leader DNA sequence comprising an intron selectedfrom the group consisting essentially of SEQ ID NO:1, SEQ ID NO:2 andSEQ ID NO:3;

(c) a structural DNA sequence that causes the production of an RNAsequence that encodes a protein; and

(d) a 3' non-translated sequence that functions in plant cells to causethe addition of polyadenylated nucleotides to the 3' end of the RNAsequence, the intron being heterologous with respect to the promoter.

Another embodiment of the invention is an isolated DNA sequenceconsisting essentially of the nucleotides shown in SEQ ID NO:1.

Another embodiment of the invention is a synthetic DNA sequence selectedfrom the group consisting essentially of the nucleotides shown in SEQ IDNO:2 and nucleotides shown in SEQ ID NO:3.

Another embodiment of the invention is transgenic plants, particularlymonocot plants, comprising the chimeric plant genes described above. Theresultant transgenic plants are capable of expressing a foreign genewhich has been inserted into the chromosome of the plant cell.

The invention provides chimeric plant genes that, when expressed in atransgenic plant, provide greater quantities of the desired proteinencoded by the structural coding sequence in the chimeric gene of theinvention. The high protein levels impart important agronomic propertiesto the plant depending on which protein is present. For example,expression of a Bacillus thuringiensis crystal toxin protein protectsthe transgenic plant from insect attack. Expression of a plant viruscoat protein protects the transgenic plant from plant viral infections.Expression of a glyphosate tolerant gene protects the transgenic plantfrom the herbicidal action of glyphosate herbicide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the DNA sequence of the intron from the 70 Kd maizeheat shock protein, SEQ ID NO:1.

FIG. 2 illustrates a truncated DNA sequence with internal deletions ofthe intron from the 70 Kd maize heat shock protein, SEQ ID NO:2.

FIG. 3 illustrates another truncated DNA sequence with internaldeletions of the intron from the 70 Kd maize heat shock protein, SEQ IDNO:3.

FIG. 4 illustrates a physical map of the plasmid pMON8677.

FIG. 5 illustrates a physical map of the plasmid pMON8678.

FIG. 6 illustrates a physical map of the plasmid pMON19425.

FIG. 7A shows the steps employed to prepare pMON19457. FIG. 7B shows thesteps employed to prepare pMON19470.

FIG. 8 illustrates a physical map of the plasmid pMON19470 comprisingthe HSP70 intron and a number of restriction sites for insertion of astructural gene encoding a protein to be expressed in plants.

FIG. 9 illustrates a physical map of the plasmid pMON19433 comprising anHSP70 intron and a GUS coding sequence.

FIG. 10 illustrates a physical map of the plasmid pMON19437 comprisingan HSP70 intron and a LUX coding sequence.

FIG. 11 illustrates a physical map of the plasmid pMON10921 comprisingan HSP70 intron and a Bt.k.-HD73 coding sequence.

FIG. 12 illustrates a physical map of the plasmid pMON19640 comprisingan HSP70 intron and an EPSPS:215 coding sequence.

FIG. 13 illustrates a physical map of the plasmid pMON19484 comprisingan HSP70 intron and a B.t.t. coding sequence.

FIG. 14 illustrates a physical map of the plasmid pMON19486 comprisingan HSP70 intron and a B.t.k.-P2 CryII coding sequence.

FIG. 15 illustrates a physical map of the plasmid pMON18131 comprisingan HSP70 intron and an ACC-deaminase coding sequence.

FIG. 16 illustrates a physical map of the plasmid pMON18103 comprising atruncated HSP70 intron and a glgC16 coding sequence.

FIG. 17 illustrates a physical map of the plasmid pMON18104 comprisingan HSP70 intron and a GOX coding sequence.

FIG. 18 illustrates a physical map of the plasmid pMON19643 comprisingthe HSP70 intron and the LUX coding sequence.

FIG. 19 illustrates a physical map of the plasmid EC9 comprising theADH1 intron.

FIG. 20 illustrates a physical map of the plasmid pMON10920 comprising aB.t.k. coding sequence--HD73 full length.

FIG. 21 illustrates a physical map of the plasmid pMON19632 comprising aADH1 intron and a GOX coding sequence.

FIG. 22 illustrates a physical map of the plasmid pMON8631 comprising amaize EPSPS coding sequence.

FIG. 23 illustrates a physical map of the cassette plasmid pMON19467comprising an HSP70 intron.

FIG. 24 illustrates a physical map of the plasmid pMON19477 comprising aBAR coding sequence.

FIG. 25 illustrates a physical map of the plasmid pMON19493 comprising aB.t.k. coding sequence--HD1/HD73 hybrid.

FIG. 26 illustrates a physical map of the plasmid pMON19648 comprising aGUS coding sequence.

FIG. 27 illustrates a physical map of the plasmid pMON19653 comprising aCP4 coding sequence.

DETAILED DESCRIPTION OF THE INVENTION

The intron of the chimeric gene of this invention was derived using thepolymerase chain reaction (PCR) from the 70 Kd maize heat shock protein(HSP70) in pMON9502 described by Rochester et al. (1986) Embo. J.,5:451-458. The intron sequence disclosed herein (SEQ ID NO:1) contains773 base pair HSP70 intron with 10 base pairs of flanking 5' exonsequence and 11 base pairs of flanking 3' exon sequence. The primersused to isolate the intron are designed such that the PCR productcontains a 6 base pair BglII site at the 5' end and a 6 base pair NcoIsite at the 3' end.

Chimeric genes are constructed by inserting the intron into BglII andNcoI sites in the 5' non-translated leader of an expression vectorcomprising a plant promoter, a scorable marker coding sequence, and apolyadenylated coding sequence. The expression vectors are constructedwith the appropriate restriction sites which permit the insertion of astructural DNA sequence encoding the desired protein. Conventionalcloning and screening procedures are used throughout unless otherwisenoted.

A gene of this invention containing the HSP70 intron can be insertedinto a suitable plant transformation vector for transformation into thedesired plant species. Suitable plant transformation vectors includethose derived from a Ti plasmid of Agrobacterium tumefaciens. A planttransformation vector preferably includes all of the necessary elementsneeded for transformation of plants or plant cells. Typical planttransformation vectors comprise selectable marker genes, one or both ofthe T-DNA borders, cloning sites, appropriate bacterial genes tofacilitate identification of transconjugates, broad host-rangereplication and mobilization functions and other elements as desired.

Transformation of plant cells may be effected by delivery of atransformation vector or of free DNA by use of a particle gun whichcomprises directing high velocity micro-projectiles coated with thevector or DNA into plant tissue. Selection of transformed plant cellsand regeneration into whole plants may be carried out using conventionalprocedures. Other transformation techniques capable of inserting DNAinto plant cells may be used, such as electroporation or chemicals thatincrease free DNA uptake.

The HSP70 intron cDNA sequence is inserted into a plant transformationvector as a gene capable of being expressed in a plant. For the purposesof this invention, a "gene" is defined as an element or combination ofelements that are capable of being expressed in a plant, either alone orin combination with other elements. Such a gene generally comprises, inthe following order, a promoter that functions in plant cells, a 5'non-translated leader sequence, a DNA sequence coding for the desiredprotein, and a 3' non-translated region that functions in plants tocause the addition of polyadenylated ribonucleotides to the 3' end ofthe mRNA transcript. In this definition, each above described element isoperationally coupled to the adjacent element. A plant gene comprisingthe above elements can be inserted by known, standard recombinant DNAmethods into a plant transformation vector and other elements added tothe vector when necessary. A plant transformation vector can be preparedthat has all of the necessary elements for plant expression except thatthe desired DNA region encoding a protein or portion thereof, which DNAcoding region can readily be added to the vector by known methods.Generally, an intron of this invention is inserted into the 5'non-translated leader sequence.

Any promoter that is known or found to cause transcription of DNA inplant cells can be used in the present invention. The amount ofenhancement of expression by use of the introns of this invention mayvary from promoter to promoter as has been observed by use of otherintrons. See Callis et al., supra, and Mascanenkas et al., supra.Suitable promoters can be obtained from a variety of sources such asplants or plant DNA viruses and include, but are not necessarily limitedto, promoters isolated from the caulimovirus group, such as thecauliflower mosaic virus 19S and 35S (CaMV19S and CaMV35S) transcriptpromoters or the figwort mosaic virus full-length transcript promoter(FMV35S). The FMV35S promoter causes a high level of uniform expressionof a protein coding region coupled thereto in most plant tissues. Otheruseful promoters include the enhanced CaMV35S promoter (eCaMV35S) asdescribed by Kat et al. (1987) Science 236:1299-1302, and the smallsubunit promoter of ribulose 1,5-bisphosphate carboxylase oxygenase(RUBISCO).

Examples of other suitable promoters are rice actin promoter;cyclophilin promoter; ubiquitin promoter; ADH1 promoter, Callis et al.,supra.; Class I patatin promoter, Bevan et al. (1986) Nucleic Acids Res.14 (11), 4675-4638; ADP glucose pyrophosphorylase promoter;β-conglycinin promoter, Tiemey et al. (1987) Planta 172: 356-363; E8promoter, Deikman et al. (1988) Embo J. 7 (11) 3315-3320; 2AII promoter,Pear et al. (1989) Plant Mol. Biol. 13: 639-651; acid chitinasepromoter, Samac et al. (1990) Plant Physiol. 93: 907-914;

The promoter selected should be capable of causing sufficient expressionof the desired protein alone, but especially when used with the HSP70intron, to result in the production of an effective amount of thedesired protein to cause the plant cells and plants regeneratedtherefrom to exhibit the properties which are phenotypically caused bythe expressed protein. In particular, the enhanced CaMV35S promoter orthe FMV35S promoter is useful in the present invention. The enhancedCaMV35S promoter causes sufficient levels of the protein mRNA sequenceto be produced in plant cells.

The mRNA produced by the promoter contains a 5' non-translated leadersequence. This non-translated leader sequence can be derived from anysuitable source and can be specifically modified to increase translationof the mRNA. The 5' non-translated region can be obtained from thepromoter selected to express the gene, the native 5' leader sequence ofthe gene or coding region to be expressed, viral RNAs, suitableeucaryotic genes, or a synthetic gene sequence. The present invention isnot limited to the construct presented in the following examples,wherein the non-translated region is derived from 45 nucleotides fromthe eCaMV35S promoter. The non-translated leader sequence can also bederived from an unrelated promoter or viral coding region as described.

The 3' non-translated region of the chimeric plant gene contains apolyadenylation signal that functions in plants to cause the addition ofpolyadenylated ribonucleotides to the 3' end of the mRNA. Examples ofsuitable 3' regions are the 3' transcribed, non-translated regionscontaining the polyadenylation signal of Agrobacterium tumor inducing(Ti) plasmid genes, such as the NOS gene, and plant genes such as thesoybean storage protein genes and the small subunit promoter of theRUBISCO gene. An example of a preferred 3' region is that from thenopaline synthase gene as described in the examples below.

In order to determine that the isolated HSP70 intron sequence includedthe desired intron region and to demonstrate the effectiveness andutility of the isolated HSP70 intron, reporter genes were inserted intoplant cassette vectors. The reporter genes chosen were the E. coliβ-glucuronidase (GUS) coding sequence and the luciferase (LUX) codingsequence.

The chimeric gene of this invention may contain any structural geneencoding a protein to be expressed in plants. An example of a suitableprotein for use in this invention is EPSP synthase(5-enolpyruvyl-3-phosphoshikimate synthase; EC:25.1.19) which is anenzyme involved in the shikimic acid pathway of plants. The shikimicacid pathway provides a precursor the the synthesis of aromatic aminoacids essential to the plant. Specifically, EPSP synthase catalyzes theconversion of phosphoenol pyruvate and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimate acid. A herbicide containingN-phosphonomethylglycine inhibits the EPSP synthase enzyme and therebyinhibits the shikimic acid pathway of the plant. The term "glyphosate"is usually used to refer to the N-phosphonomethylglycine herbicide inits acidic or anionic forms. Novel EPSP synthase enzymes have beendiscovered that exhibit an increased tolerance to glyphosate containingherbicides. In particular, an EPSP synthase enzyme having a singleglycine to alanine substitution in the highly conserved region havingthe sequence: -L-G-N-A-G-T-A- located between positions 80 and 120 inthe mature wild-type EPSP synthase amino acid sequence has been shown toexhibit an increased tolerance to glyphosate and is described in thecommonly assigned U.S. Pat. No. 4,971,908 entitled "Glyphosate-Tolerant5-Enolpyruvyl-3-Phosphoshikimate Synthase," the teachings of which arehereby incorporated by reference hereto. Methods for transforming plantsto exhibit glyphosate tolerance are discussed in the commonly assignedU.S. Pat. No. 4,940,835 entitled "Glyphosate-Resistant Plants," thedisclosure of which is specifically incorporated herein by reference. Aglyphosate-tolerant EPSP synthase plant gene encodes a polypeptide whichcontains a chloroplast transit peptide (CTP) which enables the EPSPsynthase polypeptide (or an active portion thereto) to be transportedinto a chloroplast inside the plant cell. The EPSP synthase gene istranscribed into mRNA in the nucleus and the mRNA is translated into aprecursor polypeptide (CTP/mature EPSP synthase) in the cytoplasm. Theprecursor polypeptide is transported into the chloroplast.

Another example of a suitable protein for use in this invention isglyphosate oxidoreductase (GOX) enzyme which is an enzyme which convertsglyphosate to aminomethyl-phosphorate and glyoxylate. By expressing theGOX enzyme in plants results in plants tolerant to glyphosate herbicide.The amino acid sequence of the GOX enzyme and modified genes encodingthe GOX enzyme adapted for enhanced expression in plants are describedin the commonly assigned patent application entitled "GlyphosateTolerant Plants" having U.S. Ser. No. 07/717,370 filed Jun. 24, 1991,the teachings of which are hereby incorporated herein by reference.

Other examples of suitable proteins for use in this invention areBacillus thuringiensis (B.t.) crystal toxin proteins which whenexpressed in plants protect the plants from insect infestation becausethe insect, upon eating the plant containing the B.t. toxin proteineither dies or stops feeding. B.t. toxin proteins toxic to eitherLepidopteran or Coleopteran insects may be used. Examples ofparticularly suitable DNA sequences encoding B.t. toxin protein aredescribed in the commonly assigned patent application entitled"Synthetic Plant Genes and Method for Preparation," EP patentapplication 385,962 published Sep. 5, 1990, the teachings of which arehereby incorporated herein by reference.

Another example of an enzyme suitable for use in this invention isaminocyclopropane-1-carboxylic acid (ACC) oxidase which when expressedin plants delays fruit ripening by reducing the ethylene level in planttissues.

Other examples of enzymes suitable for use in this invention areacetolactate synthase, RNase to impart male sterility, Mariani et al.(1990) Nature 347: 737-741, and wheat germ agglutenin.

Another example of an enzyme suitable for use in this invention is ADPglucose pyrophosphorylase which when expressed in plants enhances thestarch content.

All oligonucleotides are synthesized by the method of Adams et al.(1983) J. Amer. Chem. Soc. 105, 661. The nucleotide bases adenine,thymine, uracil, cytosine and guanine are represented by the letters A,T, U, C and G, respectively.

This invention is suitable for any member of the monocotyledonous(monocot) plant family including, but not limited to, maize, rice,barley, oats, wheat, sorghum, rye, sugarcane, pineapple, yams, onion,banana, coconut, dates and hops. The present invention has particularapplicability to the production of transgenic maize plants.

Any method suitable for transforming plant cells and regeneratingtransgenic plants may be used in the practice of this invention.Illustrative examples of methods suitable for regenerating transgenicplants are: corn (Fromm et al., 1990, Bio/Technology 8:833-839; andGordon-Kamm et al., 1990, The Plant Cell 2:603-618); rice (Wang et al.,1988, Plant Mol. Biol. 11:433-439) and wheat (Vasil et al., 1991,Bio/Technology 8:743-747).

The production of fertile transgenic monocotyledonous plants involvesseveral steps that together form the process. Generally, these stepscomprise 1) culturing the desired monocot tissue to be transformed toobtain suitable starting material; 2) developing suitable DNA vectorsand genes to be transferred into the monocot tissue; 3) inserting thedesired DNA into the target tissue by a suitable method; 4) plant cells;5) regenerating transgenic cells into fertile transgenic plants andproducing progeny; and 6) analyzing the transgenic plants and theirprogeny for the presence of the inserted heterologous DNA or foreigngene.

A preferred method of the present invention utilizes embryogenic calluswhich is suitable for transformation and regeneration as the startingplant material. Embryogenic callus is defined as callus which is capableof being transformed and subsequently being regenerated into mature,fertile transgenic plants. The embryogenic callus preferably has afriable Type II callus phenotype that performs well in tissue culture.Embryogenic callus may be obtained using standard procedures known tothose in the art (Armstrong, 1991, Maize Genetic Newsletter 65:92-93).Suitable maize embryogenic callus material may be obtained by isolatingimmature embryos from the maize plant 10 to 12 days after pollination.The immature embryos are then placed on solid culturing media toinitiate callus growth. The immature embryos begin to proliferate asType II callus after about one week and are thereafter suitable for usein the method of the present invention. Embryogenic callus suitable foruse in the method of the present invention may be obtained from theinitial callus formation on the immature embryos or may be from olderestablished callus cultures up to 2 years in age. It is preferred,however, that younger callus cultures be used to enhance the recovery offertile transgenic plants. Embryogenic callus that is between one weekand six months of age is preferred and embryogenic callus between oneweek and four weeks of age is most preferred. The embryogenic callus ofthe present invention is considered "primary" callus in that it hasnever been processed through or maintained as a suspension culture. Asuspension culture is defined as callus that has been broken up andplaced in a liquid solution for a period of 1 to 9 months to establish agrowing suspension culture. The embryogenic callus suitable for use inthe present invention has never been through a suspension cultureprocess or ever maintained as a suspension culture.

The preferred method of the present invention is applicable to anymonocot embryogenic callus that is capable of regenerating into maturefertile transgenic plants and does not depend on a particular genotype,inbred, hybrid or species of the monocot desired to be transformed. Itis to be understood, however, that the efficiency of the process willprobably vary depending on the culturability and transformability of theparticular plant line being used. In the present invention, a preferredmaize embryogenic callus may be obtained from an A188×B73 F₁ genotypehybrid line, or a derivative of this line, or an "Hi-II" genotype. Anygenotype that can give rise to a friable Type II callus material issuitable and will be useful in the method of the present invention. Theembryogenic callus may be initiated and maintained in any suitabletissue culture media that will promote the growth of callus of thedesired phenotype. Suitable tissue culture media are known to thoseskilled in the art of plant genetic engineering. The A188×B73 F₁ hybridline and Hi-II line have been successfully initiated, maintained andregenerated in the tissue culture media described in Table 1.

TABLE 1

N6 1-100-25 (1 L)

4.0 grams/L Chu (N₆) Basal Salts (Sigma C-1416)

1.0 ml/L Eriksson's Vitamin Mix (1000×stock made from Sigma E-1511Powder)

1.25 ml/L 0.4 mg/ml thiamine HCl

20 g/L Sucrose

1 ml/L 2, 4 D (1 MG/ML) (2, 4 D=2, 4, dichlorophenoxyacetic acid)

2.88 g/L L-proline

0.1 g/L Vitamin Free Casamino Acids (from Difco; Bacto Vitamin AssayCasamino Acids, Catalog#0288-01-2).

Adjust pH to 5.8, and add 2 g/L Gelrite or Phytagel, autoclave for 30minutes, and pour into 25×100 mm petri dishes in sterile hood.

N6 1-0-25 same as N6 1-100-25 except that no Casamino Acids are used.

N6 2-100-25 same as N6 1-100-25 above, except that 2 ml/L 2,4 D (1mg/ml) is used.

N6 2-0-0 same as N6 1-100-25 above, except 2 ml/L 2,4 D (1 mg/ml) and noVitamin Free Casamino Acids and no L-proline.

N6 6% 0 D same as N6 2-0-0 above, except 60 g/L sucrose 0 ml/L 2,4 D,and 0 L-proline is used.

MS 0.1 D 4.3 g/L MS salts (Sigma), 20 g/L sucrose, 100 mg/Lmyo-inositol, 1.3 mg/L nicotinic acid, 0.25 mg/L each of thiamine-HCL,pyridoxine and calcium pantothenate, 0.1 ml/L of 2, 4 D (1 mg/ml), 10-7MAbscisic Acid (ABA).

MS 0 D same as MS 0.1 D above, except no 2, 4 D and no ABA.

Once the desired embryogenic callus culture has been obtained,transformation of the tissue is possible. A foreign gene or genes ofinterest may be transferred to the embryogenic callus. Generally, theDNA inserted into the embryogenic callus is referred to as heterologousDNA. The heterologous DNA may contain one or more foreign genes whichmay or may not be normally present in the particular monocotyledonousplant being transformed. A foreign gene is typically a chimeric orrecombinant gene construct comprising a sequence of DNA which may or maynot be normally present in the genome of the particular monocot beingtransformed. The heterologous DNA generally contains a foreign genewhich comprises the necessary elements for expression of a desiredpolypeptide in the particular plant. Heterologous DNA suitable fortransformation into a monocotyledonous plant typically contains foreigngenes coding for a polypeptide which confers a desired trait orcharacteristic to the plant being transformed and screenable andselectable markers for determining whether the plant material has beentransformed. A typical foreign gene capable of being expressed in amonocot contains a promoter which is capable of functioning in themonocot plant, an intron, a structural DNA coding sequence encoding adesired polypeptide and a polyadenylation site region recognized inmonocotyledonous plants. A transgene is a gene or DNA sequence that hasbeen transferred into a plant or plant cell. The details of constructionof heterologous DNA vectors and/or foreign genes suitable for expressionin monocots is known to those skilled in the art of plant geneticengineering. The heterologous DNA to be transferred to the monocotembryogenic callus may be contained on a single plasmid vector or may beon different plasmids.

The heterologous DNA to be used in transforming the embryogenic callusin the method of the present invention preferably includes a selectablemarker gene which allows transformed cells to grow in the presence of ametabolic inhibitor that slows the growth of non-transformed cells. Thisgrowth advantage of the transgenic cells allows them to bedistinguished, over time, from the slower growing or non-growing cells.Alternatively, or in conjunction with a selectable marker, a visualscreenable marker such as the E. coli β-glucuronidase gene or fireflyluciferase gene (deWet et al., 1987, Mol. Cell Biol. 7:725-737) alsofacilitates the recovery of transgenic cells.

Preferred selectable marker genes for use in the method of the presentinvention include a mutant acetolactate synthase gene or cDNA whichconfers tolerance to sulfonylurea herbicides such as chlorsulfuron, theNPTII gene for resistance to the antibiotic kanamycin or G418 or a bargene for resistance to phosphinothricin or bialaphos.

The foreign gene selected for insertion into the monocot embryogeniccallus can be any foreign gene which would be useful if expressed in amonocot. Particularly useful foreign genes to be expressed in monocotsinclude genes which confer tolerance to herbicides, tolerance toinsects, tolerance to viruses, and genes which provide improved or newcharacteristics which effect the nutritional value or processingcapabilities or qualities of the plant. Examples of suitableagronomically useful genes include the insecticidal gene from Bacillusthuringiensis for conferring insect resistance and the5'-enolpyruvyl-3'-phosphoshikimate synthase (EPSPS) gene and any variantthereof for conferring tolerance to glyphosate herbicides. As is readilyunderstood by those skilled in the art, many other agronomicallyimportant genes conferring desirable traits can be introduced into theembryogenic callus in conjunction with the method of the presentinvention. One practical benefit of the technology of the presentinvention is the production of transgenic monocotyledonous plants thathave improved agronomic traits.

Once the transformation vectors containing the desired heterologous DNAhave been prepared, the DNA may be transferred to the monocotembryogenic callus through use of the microprojectile bombardmentprocess which is also referred to as particle gun technology or theBiolistics process. The heterologous DNA to be transferred is initiallycoated onto a suitable microprojectile by any of several methods knownto those skilled in the plant genetic engineering art. Themicroprojectiles are accelerated into the target embryogenic callus by amicroprojectile gun device. The design of the accelerating device or gunis not critical so long as it can perform the acceleration function. Theaccelerated microprojectiles impact upon the prepared embryogenic callusto perform the gene transfer. When the microprojectile bombardmentprocess is utilized, the DNA vector used to transfer the desired genesto the embryogenic callus is typically prepared as a plasmid vector andis coated onto tungsten or gold microprojectiles.

While any particle gun device may be used, the Biolistics PDS 1000microprojectile gun device was used in the present invention. Thisdevice had a stopping plate configuration similar to commerciallyavailable stopping plates except that the lexan disk is 3/8" thick witha 3/32" diameter hole through the disk center. The hole is enlarged atthe upper surface to 7/16" and this tapers in a countersunk arrangementto a depth of 1/4" at which point it narrows to the 3/32" diameter holewhich does not have a taper for the remaining 1/18" thickness. Theembryogenic target tissue is set at level 4 of this device which is onelevel from the bottom. The callus tissue sample was subjected to 1-3shots. A shielding metal screen with 100μ openings is typically used onthe shelf position immediately below the stopping plate. The process isperformed under a suitable vacuum.

After the embryogenic calli have been bombarded with the desiredheterologous DNA vector, the bombarded cells are grown for several daysin non-selective culturing media and then placed on a selective mediawhich inhibits the growth of the non-transformed cells, but allowstransgenic cells to continue to grow. In about 8 weeks, the continuedgrowth of the transgenic callus cells is apparent as a large growingcalli and can be recovered and individually propagated. The transgenicembryogenic callus may then be regenerated into whole, mature transgenicplants pursuant to protocols for regenerating non-transformedembryogenic callus. Generally, when regenerated plants reach thethree-leaf stage and have a well developed root system, they can betransferred to soil and hardened off in a growth chamber for two weeksbefore transfer to a greenhouse. The transformed embryogenic callus ofthe present invention respond well to regeneration procedures which workfor non-transgenic callus.

Regenerated plants may subsequently be moved to a greenhouse and treatedas normal plants for pollination and seed set. The confirmation of thetransgenic nature of the callus and regenerated plants may be performedby PCR analysis, antibiotic or herbicide resistance, enzymatic analysisand/or Southern blots to verify transformation. Progeny of theregenerated plants may be obtained and analyzed to verify thehereditability of the transgenes. This illustrates the stabletransformation and inheritance of the transgenes in the R₁ plant.

The following examples are provided to illustrate the method of thepresent invention and should not be interpreted in any way to limit thescope of the invention. Those skilled in the art will recognize thatvarious modifications can be made to the methods described herein whilenot departing from the spirit and scope of the present invention.

DESCRIPTION OF PREFERRED EMBODIMENTS Example 1

Synthesis of HSP70 Intron by Polymerase Chain Reaction

The HSP70 intron was synthesized using the polymerase chain reactionfrom a genomic clone containing a maize HSP70 gene (pMON9502: Rochesteret al., 1986, Embo J., 5:451-458).

Two different oligonucleotide primers were used in the PCR reaction. Thefirst primer consists of nucleotides 1-26 of SEQ ID NO:1 and contains aBglII site for cloning, ten nucleotides of flanking HSP70 exon 1sequence, and ten bases of the intron sequence. The second primer is thereverse complement of bases 791 through 816 of SEQ ID NO:1 and contains10 bp of intron sequence, 11 nucleotides of flanking 3' HSP70 exonsequence, and an NcoI site for cloning.

The "HSP70 intron," bases 7-812, contains the entire intron from a maizeHSP70 gene (bases 17-799) plus 10 nucleotides from HSP70 exon 1 (bases7-16) and 11 bases from HSP70 exon 2 (bases 800-812). Bases 1-6 and813-816 include restriction sites used in cloning. Base 802 was a G inthe native HSP70 exon, but has been replaced by an A for maximumenhancement of gene expression.

PCR was carried out in 100 ul reactions which contained 10 ng pMON9502DNA, 40 pmole each of SEQ7 and SEQ20, 10 mM Tris-HCL (pH8.3), 50 mM KCl,1.5 mM MgCl2, 0.01%(w/v) gelatin, 20 nmole of each dNTP, and 2.5 unitsAmplitaq DNA Polymerase (Perkin Elmer Cetus). Twenty eight cycles wererun (denaturation 1 minute at 94° C., annealed 2 minutes at 50° C., andelongated 3 minutes at 72° C. per cycle).

The PCR reaction products were were purified by phenol:chloroform (1:1)extraction, followed by digestion with BglII and NcoI. The 0.8 kb HSP70intron fragment was isolated by gel electrophoresis followed bypurification over an Elutip-D column (Schlesser & Schuell). The HSP70intron sequences were verified by the Sanger dideoxy DNA sequencingmethod. The sequence of the HSP70 intron is designated SEQ ID NO:1 andis shown in FIG. 1. The 0.8 kb HSP70 intron fragment was then clonedinto the BglII and NcoI sites within the 5' untranslated leader regionof pMON8677 to form pMON19433 as described below.

Example 2

Effect of HSP70 Intron on Corn Gene Expression in Transient Assays

A. Preparation of pMON8677, pMON8678, pMON19433, pMON19425, pMON19400,and pMON19437

pMON8677 (FIG. 4) was constructed using well characterized geneticelements. The 0.65 kb cauliflower mosaic virus (CaMV) 35S RNA promoter(e35S) containing a duplication of the -90 to -300 region (Kay et al.,1987, Science 236:1299-1302), the 1.9 kb coding sequences from the E.coli β-glucuronidase (GUS) gene (Jefferson et al., 1986, PNAS83:8447-8451) and a 0.25 kb fragment containing the 3' polyadenylationsequences from the nopaline synthase (NOS) gene (Fraley et al., 1983,Proc. Natl. Acad. Sci. 80:4803-4807) were each inserted into pUC119(Yanisch-Perron et al., 1985, Gene 33:103-119) to form the plant geneexpression vector pMON8677.

pMON8678 (FIG. 5) was formed by inserting a 0.6 kb fragment containingthe first intron from the ADH1 gene of maize (Callis et al., 1987, Genesand Dev. 1:1183-1200) into pMON8677 as described in Vasil et al. (1991)Bio/Technology 9:743-747. The monocot expression region in pMON8678 isidentical to pMON8677 except that it contains the ADH1 intron fragmentin the 5' untranslated leader.

pMON19433 (FIG. 9) was constructed by cloning the BglII-NcoI digestedPCR fragment containing the maize HSP70 intron sequences into theNcoI-BglII sites in pMON8677 to produce a monocot expression vectorequivalent to pMON8677 except that it contains the maize HSP70 intronfragment in the 5' untranslated leader.

pMON19425 (FIG. 6) was constructed by inserting the 0.65 kb cauliflowermosaic virus (CaMV) 35S RNA promoter (e35S) containing a duplication ofthe -90 to -300 region, the 1.8 kb fragment of the firefly luciferase(LUX) gene (Ow et al., 1986, Science 234:856-859; DeWet et al., 1987,Mol. Cell Biol. 7:725-737), and the 0.25 kb fragment containing the NOSpolyadenylation sequences into pUC119 (Yanisch-Perron et al., 1985,supra).

pMON19400 (FIG. 18) was formed by replacing the GUS coding sequence inpMON8678 with the 1.8 kb fragment of the LUX gene. The monocotexpression region in pMON19400 is identical to pMON19425 except that itcontains the ADH1 intron fragment in the 5' untranslated leader.

pMON19437 (FIG. 10) was constructed by cloning HSP70 intron sequencefrom pMON19433 as a 0.8 bp NcoI-BglII fragment into the NcoI-BglII sitesin pMON19425 to produce a monocot expression vector equivalent topMON19425 except that it contains the maize HSP70 intron fragment in the5' untranslated leader.

B. Analysis of Gene Expression Using Transient Assays

Two transient gene expression systems were used to evaluate expressionfrom the HSP70 intron and ADH1 intron vectors in corn cells. Two corncell lines were transformed shooting corn cells or tissues by highvelocity projectiles coated with the indicated plasmid DNA. One cellline was Black Mexican Sweet (BMS) corn, a nonregenerable corn callussuspension cells. The other cell line was BC17 corn used as tissue fromcorn leaves obtained from 4 week old plants from the innermost leaves atthe nodes around the tassel primordia.

Plasmid DNAs were prepared by using standard alkaline lysis followed byCsCl gradient purification (Maniatis et al., 1982, Molecular Cloning: ALaboratory Manual, CSH Labs). Plasmid DNA was precipitated onto tungstenM10 particles by adding 25 ul of particles (25 mg/ml in 50% glycerol), 3ul experimental plasmid DNA (1 ug/ul), 2 uL internal control plasmid DNA(1 ug/ul), 25 uL 1M calcium chloride, and 10 uL 0.1M spermidine, andvortexing briefly. The particles were allowed to settle for 20 minutes,after which 25 ul of supernatant was removed and discarded. Twoindependent particle preparations were done for each vector evaluated.

The particle preparations were then bombarded into the tissue/cells asfollows. Each sample of DNA-tungsten was sonicated briefly and 2.5 ulwas bombarded into the tissue/cells contained on one plate using aPDS-1000 (DuPont) Biolistics particle gun. Three plates of tissue/cellswere bombarded from each particle preparation.

The tissue/cells were harvested after a 24-48 hours incubation (25° C.,dark). The cells/tissues from the three bombarded plates from eachparticle preparation were combined, frozen with liquid nitrogen, andground to a fine white powder with a mortar and pestle. Each sample wasthawed on ice in 1 ml of GUS extraction buffer (GEB: 0.1M KPO4 pH7.8, 1mM EDTA, 10 mM DTT, 0.8 mM PMSF, and 5% glycerol). The samples were thenvortexed and centrifuged at 8K for 15 minutes at 5° C., and thesupernatant was transferred to a fresh tube. When enzyme assays were notperformed immediately, the samples were frozen on dry ice and stored at-80° C.

Transient β-glucuronidase gene expression was quantitated using afluorometric assay (Jefferson et al., 1987, Embo. J. 6:3901-3907). Fiftyul crude extract was assayed in one ml GEB containing 2 mM 4-methylumbelliferyl glucuronide. At 0, 10, 20, and 30 minute timepoints, 100 ulaliquots were removed and the reaction terminated by addition to 2 ml0.2M Na2CO3. Fluorescence from each sample was then determined using aHoescht DNA Fluorometer (model TKO 100). GUS activity is expressed asthe slope of fluorescence versus reaction time.

Quantitative luciferase assays were performed as follows. 50 ul ofextract was added to a cuvette containing 0.2 mls of 25 mM TricinepH7.8, 15 mM MgCl2, 5 mM ATP, and 0.5 mg/ml BSA. The 0.5 mM luciferinsubstrate was automatically dispensed by the luminometer (BertholdBioluminat LB9500C) and the peak luminescence measured during a 10second count at 25° C. Three to ten reactions were run per sample. LUXactivity is expressed as the mean light units per ul of extract.

All vectors tested were co-bombarded with internal control vectors whichencoded proteins whose enzymatic activities were distinct from those ofthe vectors being evaluated. For example, in the experiments in whichLUX vectors being evaluated, pMON8678 (GUS) was used as the internalcontrol vector, and when GUS vectors being tested pMON19400 (LUX) wasused as the internal control vector. To correct for any variability inthe procedure the results were then expressed as a ratio of theexperimental reporter gene expression to the internal control reportergene expression. The results are summarized in Table 2.

As shown in Table 2A, the HSP70 intron vectors gave significantlyincreased gene expression in BMS suspension cells when compared tovectors containing no intron (40 fold increase) or the ADH1 intron (4fold increase) vectors. This effect was observed using either GUS or orLUX as the reporter gene. Table 2B shows that this effect is not limitedto the BMS cell system. In the leaf transient gene expression assays,the HSP70 intron vector showed an 8.7 times GUS expression level overthe control containing no intron, whereas, the ADH1 intron showed only a1.6 times GUS expression level over the control containing no intron.

                  TABLE 2    ______________________________________    Effects of Introns on Gene Expression    in Transient Assays    Intron    Relative GUS (vector)                             Relative LUX (vector)    ______________________________________    A. Effect of introns on transient gene expression in BMS cells.    no intron  1X (pMON8677)  1X (pMON19425)    ADH1       4X (pMON8678)  4X (pMON19400)    HSP70     40X (pMON19433)                             40X (pMON19437)    B. Effect of introns on transient gene expression in maize    leaf tissue.    no intron   1X (pMON8677)    ADH1      1.6X (pMON8678)    HSP70     8.7X (pMON19433)    ______________________________________

Example 3

Effect of HSP70 Intron on Gene Expression in Stably TransformedNonregenerable Corn Cultures

A. Production of stably transformed BMS cell lines

Black Mexican Sweet corn suspension cells were transformed by particlegun bombardment essentially as described above. Plasmid DNA forbombardment was prepared and precipitated onto tungsten M10 particles byadding 12.5 ul of particles (25 mg/ml in 50% glycerol), 2.5 ul plasmidDNA (1 ug/ul), 12.5 uL 1M calcium chloride, and 5 uL 0.1M spermidine,and vortexing briefly. The particles were allowed to settle for 20minutes, after which 12.5 ul of supernatant was removed and discarded.Each sample of DNA-tungsten was sonicated briefly and 2.5 ul wasbombarded into the embryogenic cultures using a PDS-1000 biolisiticsparticle gun (DuPont). EC9 (FIG. 19), a plasmid containing anacetolactate synthase gene, was included for use in chlorsulfuronselection for transformed control cells. A second plasmid containing thetest construct was co-precipitated with EC9. BMS cells were plated onfilters and bombarded using a PDS-1000 (DuPont) particle gun. Afterbombardment, the cells were transferred to MS liquid medium for 1 dayand then plated onto solid medium containing 20 ppb chlorsulfuron. Afterapproximately 4 weeks, chlorsulfuron resistant calli were selected andgrown up for analysis of gene expression.

B. Effect of the HSP70 Intron on GUS Expression

Plasmids containing the GUS gene and no intron (pMON8677), ADH1 intron(pMON8678), or HSP70 intron (pMON19433) were bombarded into BMS cellsand stably transformed lines were produced as described above.Chlorsulfuron resistant lines were selected and then scored for GUSexpression by histochemical staining (Jefferson et al., 1987, Embo. J.6:3901-3907). As shown in Table 3A, the transformations with the HSP70intron vector showed a significantly higher proportion of co-expressionof the unselected GUS marker than did the transformation with either thevector containing the ADH1 intron or no intron. Since more chlorsulfuronresistant calli were above the threshold of detection histochemical GUSstaining, it is likely that the HSP70 intron vectors express at higherlevels than the ADH1 or no intron vectors. To confirm this, GUS activitywas quantitated in extracts from ten independent GUS positivetransformants from each vector (for pMON8677, the one GUS positivecallus was assayed; nine others were chosen randomly). The data fromthese assays is shown in Table 3B. These results indicate that the HSP70intron enhances GUS expression in stably transformed cell lines to aneven greater extent than was observed in transient gene expressionanalyses. The mean level of GUS expression observed with the linescontaining the HSP70 intron vector was approximately 80 fold over thatobserved in lines containing the ADH1 intron vector. The best of the tenHSP70 lines expresses over 100 fold more GUS than the best ADH1 line andapproximately 800 fold over the best line without an intron.

                  TABLE 3    ______________________________________    Effect of Introns on GUS Expression    in Stable Transformants    ______________________________________    A. GUS expressing BMS calli - number and percentage.    ______________________________________    Class*     pMON8677   pMON8678   pMON19433    ______________________________________               No Intron  ADHl intron                                     HSP70 intron    -          79 (99%)   48 (67%)   28 (47%)    +           0 (0%)    14 (19%)    2 (3%)    ++          1 (1%)     9 (13%)    7 (12%)    +++         0 (0%)     1 (1%)    22 (37%)               80         72         59    ______________________________________    B. Levels of GUS expression in BMS calli.    ______________________________________    Vector     Intron     Range**    Mean**    ______________________________________    pMON8677   none       0-38       N.D.    pMON8678   ADH1       28-219     95 + 75    pMON19433  HSP70      1594-29,629                                     7319 ± 9016    ______________________________________     *     - no cells show expression     + a few cells show GUS expression     ++ some cells show GUS expression     +++ all cells show strong GUS expression     **(pmol/min/mg)

Example 4

Effect of HSP70 Intron on B.t.k. Expression in Stably Transformed BMSCell Lines

We have similarly examined the effect of the HSP70 intron on expressionof the commercially important B.t.k. gene. Two plasmids were constructedthat only differed by the intron they contained: pMON10920(e35S/ADH1/B.t.k./NOS) and pMON10921 (e35S/HSP70/B.t.k./NOS). Eachcontained a 3.6 kb fully synthetic gene encoding the Bacillusthuringiensis kurstaki (B.t.k.) insect control protein described byAdang et al. (1985) Gene 36: 289-300. Expression of this gene in plantsresults in insect resistance. pMON10920 (FIG. 20) was constructed byinserting the 3.6 kb NcoI/EcoRI fragment containing the B.t.k. intopMON8678 (FIG. 5), replacing the 1.9 kb GUS fragment. pMON10921 (FIG.11) was constructed similarly, except that the 3.6 kb NcoI/EcoRIfragment containing the B.t.k. coding sequence was inserted intopMON19433 (FIG. 9).

BMS lines were co-transformed with each of these plasmids and EC9 (ALS)as described in Example 3A. Approximately thirty independentchlorsulfuron resistant lines were generated in each transformation.These calli were tested for Tobacco Hornworm (THW) toxicity, and theinsect resistant lines were assayed further. The amount of B.t.k.protein in soluble extracts from each THW resistant callus was measuredby ELISA and expressed as a percentage of total protein. Of the 11insect positive lines containing the ADH1 intron vector (pMON10920),only one line contained enough B.t.k. protein to be detected in theELISA assay. The amount was 0.4×10⁻⁵ %. Twenty of the 29 THW resistantlines containing the HSP70 intron vector (pMON10921) produced enoughprotein for detection by ELISA. The average amount was 5.1×10⁻⁵ % with arange of <0.01-10.5×⁻⁵ %. When the mean B.t.k. protein levels arecompared, the HSP70 intron vector increases expression 12 fold over theADH1 intron vector.

Example 5

Effect of HSP70 Intron on GOX Expression in BMS Transformants

pMON19632 and pMON 19643 were constructed to examine the effects ofintrons on GOX expression. Both vectors contain a gene fusion composedof the N-terminal 0.26 Kb chloroplast transit peptide sequence derivedfrom the Arabidopsis thaliana SSU 1a gene (SSU CTP) (Timko et al., 1988,The Impact of Chemistry on Biotechnology, ACS Books, 279-295) and theC-terminal 1.3 Kb synthetic GOX gene sequence. The GOX gene encodes theenzyme glyphosate oxidoreductase which catalyzes the conversion ofglyphosate to herbicidally inactive products, aminomethylphosphonate andglyoxylate. Plant expression of the gene fusion produces a pre-proteinwhich is rapidly imported into chloroplasts where the CTP is cleaved anddegraded releasing the mature GOX protein (della-Cioppa et al., 1986,Proc. Natl. Acad. Sci. USA 83: 6873-6877).

pMON19632 (FIG. 22) was constructed in the same manner as pMON8678 byinserting the SSU·CTP--GOX fusion as a 1.6 kb BglII-EcoRI fragmentbetween the ADH1 intron and NOS polyadenylation sequences. Thus,pMON19632 is comprised of, from 5' to 3', the enhanced CaMV35S promoter,ADH1 intron, SSU·CTP--GOX coding sequence, and nopaline synthasepolyadenylation region in a pUC backbone containing an β-lactamase genefor ampicillin selection in bacteria.

A cassette vector pMON19470 was constructed for cloning coding sequencessuch as GOX adjacent to the HSP70 intron (FIG. 7). A receptor plasmidpMON19453 was made by inserting annealed synthetic oligonucleotidescontaining the sitesKpnI/NotI/HincII/HindIII/BglII/DraI/XbaI/NcoI/BamHI/EcoRI/EcoRV/XmaI/NotI/SacIinto PBSKS+ (Stratagene) which had been digested with KpnI and SacI. Thenopaline synthase (NOS) polyadenylation region (Fraley et al., 1983,Proc. Natl. Acad. Sci. 80:4803-4807) was inserted by digesting pMON8678(FIG. 5) with BamHI, followed filling Klenow Polymerase to create bluntends, and digesting with EcoRI. The 0.25 kb NOS fragment was insertedinto the polylinker of pMON19453 at the EcoRV/EcoRI sites to formpMON19459. pMON19457 was constructed by inserting a 0.65 kb fragmentcontaining the CaMV E35S promoter (Kay et al., 1987, Science236:1299-1302) into the HindIII/BglII sites in pMON19459. pMON19433 waslinearized with NcoI, blunt-ended with mung bean nuclease, and Xbalinkers were added. The HSP70 intron fragment was then removed bydigestion with BglII and inserted into the XbaI/BglII sites in pMON19457to form pMON19458. Synthetic linkers to change the order of therestriction sites were then inserted into pMON19458 to form pMON19467.The NotI expression cassette was removed from pMON19467 and insertedinto a pUC-like vector pMON10081 which contains the NPTII sequences frompKC7 (Rao and Rogers, 1978, Gene 3:247) to form pMON19470 (FIG. 8).Thus, pMON19470 is comprised of, from 5' to 3', the enhanced CaMV35Spromoter, HSP70 intron, polylinker for cloning coding sequences, and NOSpolyadenylation region in a pUC-like backbone containing an NPTII genefor kanamycin selection in bacteria.

pMON19643 (FIG. 17) was constructed by inserting the SSU·CTP--GOX fusioncoding sequences into pMON19470 as a 1.6 kb BglII/EcoRI fragment intoBamHI-EcoRI digested pMON19470 (FIG. 8). Thus, pMON19643 is comprisedof, from 5' to 3', the enhanced CaMV35S promoter, HSP70 intron,SSU·CTP--GOX coding sequence, and nopaline synthase polyadenylationregion in a pUC-like backbone containing an NPTII gene for kanamycinselection in bacteria.

BMS suspension cells were bombarded with pMON19632 or pMON19643 asdescribed in Example 3A. Plasmid EC9 was included in each bombardment sothat the transformed BMS cells could be selected on chlorsulfuron. Thechlorsulfuron resistant calli were transferred to 5 mM glyphosate mediumand moved to fresh 5 mM glyphosate medium after two weeks. After twoweeks, the percentage of the calli that survived on the glyphosatemedium were scored.

The results are shown in Table 4. The ADH1 intron vector (pMON19632)gave little or no glyphosate resistant calli. The HSP70 intron vector(pMON19643) showed over 40% of the chlorsulfuron resistant calli werealso resistant to glyphosate. The levels of GOX protein accumulation inthe chlorsulfuron resistant lines were measured by Western blotanalysis. As shown in Table 3, the HSP70 intron vector gave demonstrablyhigher levels of GOX expression than the ADH1 intron vector.

                  TABLE 4    ______________________________________    Effect of Introns on GOX Gene Expression    in BMS Transformants    Vector     Intron  % glp resistant                                    % GOX protein    ______________________________________    pMON19632  ADH1     2%          (0.02-0.04%)    pMON19643  HSP70   42%          (0.05-0.5%)    ______________________________________

Example 6

Effect of HSP70 Intron on EPSP Synthase and Glyphosate Selection

Two vectors, pMON8631 and pMON19640, were contructed to compare theeffects of the ADH1 and HSP70 intron on the expression of the5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene. pMON8631 (FIG.23) was constructed similarly to pMON8678 (FIG. 5), except that a 1.75kb fragment containing the maize EPSPS coding sequence with twomutations that confer tolerance the the herbicide glyphosate (Gly101>Alaand Gly163>Asp of mature peptide) was inserted between the ADH1 intronand the NOS polyadenylation sequences. Thus, pMON8631 is comprised of,from 5' to 3', the enhanced CaMV35S promoter, ADH1 intron, EPSPS codingsequence, and nopaline synthase polyadenlyation region in a pUC backbonecontaining a 13-lactamase gene for ampicillin selection in bacteria.

To form pMON19640 (FIG. 12), the 1.75 kb XbaI-EcoRI fragment frompMON8631 was inserted into the corresponding restriction sites inpMON19470 (FIG. 8). Thus, pMON19640 is comprised of, from 5' to 3', theenhanced CaMV35S promoter, HSP70 intron, EPSPS coding sequence, andnopaline synthase polyadenylation region in a pUC-like backbonecontaining an NPTII gene for kanamycin selection in bacteria.

Stably transformed BMS lines were produced by direct selection onglyphosate containing medium. Cells were bombarded with either pMON8631or pMON19640 as in described in Example 3A. After bombardment, the cellswere resuspended in MS medium without selection for one day. Glyphosatewas then added to the liquid medium to a final concentration of 5 mM,and the cultures incubated for four days. Five days post-bombardment,the cells were embedded in agarose containing 5 mM glyphosate.Approximately 6 weeks after embedding, the number of glyphosateresistant calli were scored. pMON8631 (ADH1 intron) produced 59glyphosate resistant calli, while pMON19640 (HSP70 intron) produced 117glyphosate resistant calli, a two fold increase. Although the levels ofEPSPS expression in these calli was not quantitated, it is likely thatthe HSP70 intron vector expresses more EPSPS which in turn results inmore transformation events that produce enough EPSPS to overcome thetoxic effects of the glyphosate in the medium, thus giving a higherfrequency of recovery of glyphosate resistant calli.

Example 7

Expression of Other Coding Sequences Using HSP70 Intron Vectors EncodingInsecticidal Proteins

pMON19484 (FIG. 13) containing a synthetic gene encoding the Bacillusthuringensis var. tenebrionis (B.t.t.) insecticidal protein (McPhersonet al., 1988, Bio/Technology 6: 61-66) was constructed by inserting the1.8 kb B.t.t. gene on a BglII fragment into the BamHI site in pMON19470(FIG. 8). Thus, pMON19484 is comprised of, from 5' to 3', the enhancedCaMV35S promoter, HSP70 intron, B.t.t. coding sequence, and nopalinesynthase polyadenylation region in a pUC-like backbone containing anNPTII gene for kanamycin selection in bacteria.

Stably transformed BMS calli were produced using particle gunbombardment to introduce pMON19484 as described in Example 3A. pMON19484was bombarded in combination with EC9 (FIG. 19) into BMS cells.Resistant calli were selected on 20 ppb chlorsulfuron. The resistantcalli were then assayed for expression of the B.t.t. gene.

Chlorsulfuron resistant calli bombarded with pMON19484 were screened forexpression of the B.t.t. protein utilizing a Colorado Potato Beetle(CPB) feeding assay. CPB larvae were applied to BMS callus which hadbeen blotted slightly to remove excess moisture. Five larvae wereallowed to feed on callus representing each chlorsulfuron resistantline. The level of insect mortality and/or stunting was assessed fivedays later. Forty calli were assayed. Eight calli (20%) showedinsecticidal activity, 11 calli (28%) caused stunting, 6 calli (15%)caused small amounts of stunting, and 15 calli (38%) had no effect onthe CPB insects.

The calli that showed the greatest insecticidal/stunting effects wereanalyzed further by Western blot analysis. BMS calli were dried on aWhatman filter and then extracted directly in SDS-PAGE buffer (Laemmli,1970, Nature 227: 680-685). Levels of total protein were determined(Biorad) and 40-50 ug protein loaded on a 12% SDS-PAGE gel. E.coli-produced B.t.t. protein was also loaded as quantitation standards.After gel electrophoresis, proteins were electrophoretically transferredfrom the gel to membranes (Towbin et al., 1979, PNAS 76:4350-4354). Themembranes were then incubated with an anti-B.t.t. antibody, followed bydetection using a chemiluminescent (Amersham) detection system.

Seven lines were examined. One line showed high levels of proteinexpression (0.02% total protein), four lines showed moderate B.t.t.protein levels (0.001%), and two lines did not produce enough B.t.t.protein for detection by Western blot.

pMON19486 (FIG. 14) contains a synthetic gene encoding the Bacillusthuringensis kurstaki CryIIA gene. The amino acid sequence of this gene(1.9 kb) is identical to the gene referred to as the CryB1 in Widner etal. (1989) J. Bacteriol. 171:965-974. It has insecticidal activityagainst both lepidopteran and dipteran insects. pMON19486 wasconstructed by inserting the 1.9 kb CryIIA coding sequence on a BglIIfragment into the BamHI site in pMON19470 (FIG. 8). Thus, pMON19486 iscomprised of, from 5' to 3', the enhanced CaMV35S promoter, HSP70intron, CryIIA coding sequence, and nopaline synthase polyadenylationregion in a pUC-like backbone containing an NPTII gene for kanamycinselection in bacteria.

Stably transformed BMS calli were produced using particle gunbombardment to introduce pMON19486 as described in Example 3A. pMON19484was bombarded in combination with EC9 (FIG. 19) into BMS cells.Resistant calli were selected on 20 ppb chlorsulfuron. The resistantcalli were then assayed for expression of the CryIIA gene.

Expression of the B.t.k. CryIIA protein in the chlorsulfuron resistantcalli bombarded with pMON19486 was initially detected by insecticidalactivity in a feeding assay with the sensitive Tobacco Hornworm (THW).Calli with CryIIA expression high enough to kill the THW insects werebulked up and assayed in European Corn Borer (ECB) and Fall Army Worm(FAW) insect feeding assays. Sixteen ECB or 12 FW insects werepre-weighed and then reared on the BMS calli for 7 days. The number ofsurvivors were scored to determine the degree of mortality. The amountof stunting was measured by determining the average weight gain of thesurviving insects relative to controls. The data are shown in Table 5.

Calli with insecticidal activity were also assayed for accumulation ofthe CryIIA protein by Western blot analysis as described above. Theamount of CryIIA protein was quantitated relative to E. coli producedstandards on the same blot. As shown in Table 5, six of the seveninsecticidal lines demontrated sufficient expression of the CryIIAprotein to detect by the less sensitive Western blot. The CryIIAexpression ranged from 0.004 to 0.15%, with an average of 0.007%, oftotal cellular protein.

                  TABLE 5    ______________________________________    Expression of CryIIA in Stable    BMS Transformants                        mean weight gain           # survivors/initial                        per surviving insect (mg)                                       CryIIA    Line   ECB     FAW      ECB     FAW    protein (%)    ______________________________________    control           10/16   10/12    3.0     3.9    0    12-9    0/16   10/12    all dead                                    0.5    0.004     3-20   2/16   11/12    0.8     1.5    0.004    11-31   1/16   10/12    3.7     1.2    0.015     3-4    0/16   11/12    all dead                                    0.6    0.013     3-10   1/16   12/12    3.5     0.8    0     3-38   0/16   11/12    all dead                                    0.4    0.0025     3-34   0/16   11/12    all dead                                    0.5    0.0025    ______________________________________

Example 8

pMON18103 (FIG. 16) contains a gene fusion composed of the N-terminal0.26 Kb chloroplast transit peptide sequence derived from theArabidopsis thaliana SSU 1a gene (SSU·CTP) (Timko et al., 1988, TheImpact of Chemistry on Biotechnology, ACS Books, 279-295) and the E.coli ADPglucose pyrophosphorylase mutant gene glgC16 (Leung et al.,1986, J. Bacteriol. 167: 82-88). Expression of the SSU·CTP/glgC16 fusionresults in increased starch accumulation in plant cells. pMON18103 wasconstructed by inserting the SSU·CTP/glgC16 coding sequence on a 1.6 kbXbaI fragment into the XbaI site in pMON19467 (see FIG. 23). Thus,pMON19486 is comprised of, from 5' to 3', the enhanced CaMV35S promoter,HSP70 intron, SSU·CTP/glgC16 coding sequences, and nopaline synthasepolyadenlyation region in a pUC backbone containing a β-lactamase genefor ampicillin selection in bacteria.

Stably transformed BMS calli were produced using particle gunbombardment to introduce pMON18103 as described in Example 3A. pMON19103was bombarded in combination with EC9 (FIG. 19) into BMS cells.Resistant calli were selected on 20 ppb chlorsulfuron. The resistantcalli were then assayed for expression of the glgC16 gene.

Chlorsulfuron resistant BMS lines that had been bombarded with pMON18103were assayed for starch accumulation using I₂ /IKI staining (Coe et al.,1988, in Corn and Corn Improvement, eds. GF Sprague and JW Dudley. AGSInc., Madison, Wisc. pp. 81-258). Eight of 67 lines showed increasedlevels of starch staining relative to control calli. Western blotanalyses were performed on these lines as described above. All linesshowed ADP-GPP expression, with levels from 0.02-0.1% of total proteinrelative to quantitation standards using E. coli-produced ADP-GPPprotein.

Example 9

pMON18131 (FIG. 15) contains the ACC deaminase gene from Pseudomonas.The ACC deaminase enzyme converts 1-aminocyclopropane-1-carboxylic acid(ACC) to alphaketobutyrate and ammonia (Honma and Shimomura, 1978,Agric. Biol. Chem. Vol.42 No.10: 1825-1813). The expression of the ACCdeaminase enzyme in plants results in inhibition of the ethylenebiosynthesis (Klee et al., 1991, Plant Cell Vol. 3, pp. 1187-1193) whichaffects ripening. pMON18131 was constructed by inserting the 1.1 kb ACCdeaminase gene as an Xbal-BamHI fragment into pMON18103 (FIG. 16),replacing the glgC16 coding sequence. Thus, pMON19486 is comprised of,from 5' to 3', the enhanced CaMV35S promoter, HSP70 intron, ACCdeaminase coding sequences, and nopaline synthase polyadenlyation regionin a pUC backbone containing a β-lactamase gene for ampicillin selectionin bacteria.

Stably transformed BMS calli were produced using particle gunbombardment to introduce pMON18131 as described in Example 3A. pMON18131was bombarded in combination with EC9 (FIG. 19) into BMS cells.Resistant calli were selected on 20 ppb chlorsulfuron. The resistantcalli were then assayed for expression of the ACC deaminase gene.

Chlorsulfuron resistant calli bombarded with pMON18131 were assayed byWestern blot analysis. Seventeen of 24 lines examined showed high levelsof ACC deaminase protein accumulation (˜0.1% of total protein).

Example 10

Production of Plants Using Vectors Containing the HSP70 Intron andBialaphos Selection

pMON19477 (FIG. 24) contains the BAR gene from S. hygroscopicus. The BARgene encodes a phosphinothricin acetyltransferase enzyme that can beused as a selectable marker by conferring resistance to bialaphos orphosphinothricin, the active ingredient in the herbicide BASTA (Fromm etal., 1990, Bio/Technology 8:833-839; De Block et al., 1987, Embo. J.6:2513-2518; Thompson et al., 1987, Embo. J. 6:2519-2523). pMON19477 wasconstructed by inserting the BAR gene as a 0.6 kb BamHI-BClI fragmentinto the BamHI site in pMON19470 (FIG. 8). Thus, pMON19477 is comprisedof, from 5' to 3', the enhanced CaMV35S promoter, HSP70 intron, BARcoding sequence, and nopaline synthase polyadenylation region in apUC-like backbone containing an NPTII gene for kanamycin selection inbacteria.

pMON19493 (FIG. 25) contains a "synthetic" B.t.k. gene consisting of 1.8kb truncated gene encoding amino acids 1 to 615 of the Bacillusthuringensus kurstaki CryIA(b) insect control protein described byFischhoff et al. (1987) Bio/Technology 5: 807-813, translationally fusedto the 1.8 kb 3' half of the CryIA(c) gene encoding amino acids 616-1177(Adang et al. 1985, Gene 36: 289-300). Expression of the gene in plantsresults in insect resistance. pMON19493 was constructed by inserting the3.6 kb "synthetic" B.t.k. gene coding sequence as a BglII fragment intothe BamHI site in pMON19470 (FIG. 8). Thus, pMON19493 is comprised of,from 5' to 3', the enhanced CaM 35S promoter, HSP70 intron, "synthetic"B.t.k. coding sequence, and nopaline synthase polyadenylation region ina pUC-like backbone containing an NPTII gene for kanamycin selection inbacteria.

pMON19468 (FIG. 26) contains the E. coli GUS gene and can be used as avisible scoreable marker of transformation using histochemical staining.pMON19468 was constructed using the 1.8 kb BglII-EcoRI fragmentcontaining the GUS gene from pMON8678 inserted into the BamHI-EcoRI sitein the pMON19470 backbone. Thus, pMON19468 is comprised of, from 5' to3', the enhanced CaMV35S promoter, HSP70 intron, GUS coding sequence,and nopaline synthase polyadenylation region in a pUC-like backbonecontaining an NPTII gene for kanamycin selection in bacteria.

Embryogenic cultures were initiated from immature maize embryos of the"Hi-II" genotype (Armstrong et al., 1991, Maize Genetic Newsletter65:92-93) cultured 18-33 days on N6 2-100-25-Ag medium (Chu et al.,1975, Sci. Sin. Peking 18:659-688) modified to contain 2 mg/L2,4-dichlorophenoxyacetic acid, 180 mg/L casein hydrolysate, 25 mML-proline, 10 uM silver nitrate, pH5.8, solidified with 0.2% Phytagel™(Sigma). These embryogenic cultures were used as target tissue fortransformation by particle gun bombardment.

A 2:1:1 mixture of pMON19477, pMON19493, and pMON19468 plasmid DNAs wasprecipitated onto tungsten M10 particles by adding 12.5 ul of particles(25 mg/ml in 50% glycerol), 2.5 ul experimental plasmid DNA (1 ug/ul),12.5 ul 1M calcium chloride, and 5 ul 0.1M spermidine, and vortexingbriefly. The particles were allowed to settle for 20 minutes, afterwhich 12.5 ul of supernatant was removed and discarded. Each sample ofDNA-tungsten was sonicated briefly and 2.5 ul was bombarded into theembryogenic cultures using a PDS-1000 Biolisitics particle gun (DuPont).

The tissue was transferred to fresh, nonselective medium the day afterbombardment. Six days post-bombardment, the material was transferred toselective media containing 2 mg/L 2,4-dichlorophenoxyacetic acid, 10 uMsilver nitrate, no casamino acids or proline, and 0.3 mg/L bialaphos.After 2-3 weeks, the cultures were transferred to fresh media whichcontained 1.0 mg/L bialaphos. The cultures were maintained on the 1.0mg/L bialophos media, transferred at 2-3 week intervals, untilbialaphos-resistant calli could be distinguished. Seven bialaphosresistant calli were recovered from eight plates of embryogenicmaterial.

Bialaphos resistant lines were bulked up and assayed for B.t.k. or GUSexpression. All lines were tested for insecticidal activity in TobaccoHornworm (THW) feeding assays to test for B.t.k. expression.Approximately 0.5 g of the embryogenic callus was fed to 10-12 THWlarvae. Two lines, 284-5-31 and 284-6-41, were positive and showedsignificant lethality to the THW insects, indicating that the B.t.k.gene from pMON19493 had integrated into their genomes and was beingexpressed. All lines were also assayed for GUS expression using ahistochemical assay (Jefferson, R. A., Kavanagh, T. A., and Bevan, M.W., 1987, Embo. J. 6:3901-3907). Of the seven lines tested, only asingle line, 284-8-31, showed any detectable blue staining indicative ofGUS expression from pMON19468.

Plants were regenerated from all of the bialophos resistant calli in athree step regeneration protocol. All regeneration was performed on 1mg/L BASTA. Embryogenic tissue was incubated on each medium for abouttwo weeks and then transferred to the medium for the next step (seeTable 6 for regeneration media ingredients). The first two steps werecarried out in the dark at 28° C., and the finalstep under a 16:8 hourphotoperiod, ˜70 uE m-2 sec-1 provided by cool-white fluorescent bulbs,at ˜25° C. Small green shoots that formed on Regeneration Medium 3 in100×25 mm Petri plates are transferred to Regeneration Medium 3 in200×25 mm Pyrex™ or Phytatrays™ to permit further plantlet developmentand root formation. Upon formation of a sufficient root system, theplants were carefully removed from the medium, the root system washedunder running water, and the plants placed into 2.5" pots containingMetromix 350 growing medium. The plants were maintained for several daysin a high humidity environment, and then the humidity was graduallyreduced to harden off the plants. The plants were transplanted from the2.5" pots to 6" pots and finally to 10" pots during growth.

                  TABLE 6    ______________________________________    Regen 1     Regen 2        Regen 3    ______________________________________    MS salts (Sigma;                N6 salts (Sigma;                               MS salts (Sigma;    4.4 g/L     4.0 g/L        4.4 g/L    1.30 mg/L nicotinic                0.5 mg/L nicotinic                               1.30 mg/L nicotinic    acid        acid           acid    0.25 mg/L pyridoxine                0.5 mg/L pyridoxine                               0.25 mg/L pyridoxine    HC1         HC1            HC1    0.25 mg/L thiamine                1.0 mg/L thiamine                               0.25 mg/L thiamine    HC1         HC1            H1    0.25 mg/L   2.0 mg/L glycine                               0.25 mg/L    Ca-pantothenate            Ca-pantothenate    100 mg/L myo-inositol                60 g/L sucrose 100 mg/L myo-inositol    1 mM asparagine                2.0 g/L Phytagel ™                               1 mM asparagine    0.1 mg/L 2,4-D                pH 5.8         20 g/L sucrose    0.1 μM ABA              2.0 g/L Phytagel ™    20 g/L sucrose             pH 5.8    2.0 g/L Phytagel ™    pH 5.8    ______________________________________     All ingredients can be obtained from Sigma Chemical Co., P.0. Box 14508,     St. Louis, MO 63178.

All corn plants regenerated from bialaphos resistant embryogenic calliwere shown to express at least one of the genes that had been bombarded:BAR, B.t.k., or GUS. Plants regenerated from the bialophos resistant,THW negative callus lines were confirmed to be transgenic and expressingthe BAR gene by BASTA leaf painting assays. Seedlings were assayed when4-5 leaves had fully emerged from the whorl. A solution of 1% BASTA,0.1% Tween20 was applied to the upper and lower surfaces of the firstfully emerged leaf. The plants were scored three days after painting.The control plants showed yellowing and necrosis on the leaves, whilethe leaves from the resistant lines were green and healthy. Thisindicates not only that the BAR gene in pMON19477 was expressed in theseplants, but also that the expression levels were high enough to conferresistance to the herbicide BASTA at the plant level.

Plants regenerated from the two lines that had shown THW activity,284-5-31 and 284-6-41, were assayed for B.t.k. expression by whole plantfeeding assays. Plants approximately 30" in height were inoculated with100 European Corn Borer (ECB) eggs. Feeding damage was scored on a scaleof 0 (no damage) to 9 (high level of leaf feeding damage) two weeksafter inoculation. The control plants scored insect feeding ratings of9. All plants from either line containing pMON19493 received ratings ofzero; no ECB damage was present.

The ECB feeding studies indicate that the B.t.k. gene was expressed athigh enough levels in the regenerated plants to impart insectresistance. To quantitate the level of expression, samples from theregenerated lines were assayed by ELISA. Eight plants regenerated fromeach callus line were analyzed. Plants from line 284-5-31 ranged inB.t.k. expression from 0.006 to 0.034% of total cellular protein, withan average value of 0.02%. Plants from line 284-6-41 ranged in B.t.k.expression from 0.005 to 0.05%, also with an average of 0.02% of totalprotein.

Example 11

Production of Plants Using Glyphosate Selection Vectors Containing theHSP70 Intron

pMON19640 (FIG. 12) contains a 5-enolpyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. To form pMON19640 (FIG. 12), a 1.75 kb XbaI-EcoRIfragment containing the maize EPSPS coding sequence with two mutations(Gly144>Ala and Gly206>Asp) of mature peptide that confers tolerance toglyphosate herbicide was inserted into the corresponding restrictionsites in pMON19470 (FIG. 8). Thus, pMON19640 is comprised of, from 5' to3', the enhanced CaMV35S promoter, HSP70 intron, EPSPS coding sequence,and nopaline synthase polyadenylation region in a pUC-like backbonecontaining an NPTII gene for kanamycin selection in bacteria.

pMON19643 (FIG. 17) contains a gene fusion composed of the N-terminal0.26 Kb chloroplast transit peptide sequence derived from theArabidopsis thaliana SSU 1a gene (SSU CTP) (Timko et al., 1988, TheImpact of Chemistry on Biotechnology, ACS Books, 279-295) and theC-terminal 1.3 Kb synthetic GOX gene sequence. The GOX gene encodes theenzyme glyphosate oxidoreductase which catalyzes the conversion ofglyphosate to herbicidally inactive products, aminomethylphosphonate andglyoxylate. Plant expression of the gene fusion produces a pre-proteinwhich is rapidly imported into chloroplasts where the CTP is cleaved anddegraded releasing the mature GOX protein (della-Cioppa et al., 1986,Proc. Natl. Acad. Sci. USA 83: 6873-6877). pMON19643 (FIG. 18) wasconstructed by inserting the SSU·CTP--GOX fusion coding sequences intopMON19470 as a 1.6 kb BglII/EcoRI fragment into BamHI-EcoRI digestedpMON19470 (FIG. 8). Thus, pMON19643 is comprised of, the from 5' to 3',enhanced CaMV35S promoter, HSP70 intron, SSU·CTP--GOX coding sequence,and nopaline synthase polyadenylation region in a pUC-like backbonecontaining an NPTII gene for kanamycin selection in bacteria.

Embryogenic cultures were initiated from immature maize embryos of the"Hi-II" genotype (Armstrong et al., 1991, Maize Genetic Newsletter65:92-93) cultured 18-33 days on N6 1-100-25 medium (Chu et al., 1975,Sci. Sin. Peking, 18:659-688) modified to contain 1 mg/L2,4-dichlorophenoxyacetic acid, 180 mg/L casein hydrolysate, 25 mML-proline, and solidified with 0.2% Phytagel™ (Sigma). These embryogeniccultures were used as target tissue for transformation by particle gunbombardment.

A 1:1 mixture of PMON19640 and pMON19643 plasmid DNAs was precipitatedonto tungsten M10 particles by adding 12.5 ul of particles (25 mg/ml in50% glycerol), 2.5 ul experimental plasmid DNA (1 ug/ul), 12.5 ul 1Mcalcium chloride, and 5 ul 0.1M spermidine, and vortexing briefly. Theparticles were allowed to settle for 20 minutes, after which 12.5 ul ofsupernatant was removed and discarded. Each sample of DNA-tungsten wassonicated briefly and 2.5 ul was bombarded into the embryogenic culturesusing a PDS-1000 Biolistics particle gun (DuPont).

One week after bombardment, cultures were transferred to fresh N6 1-0-25medium (same as the initiation medium, except removing caseinhydrolysate and adding 1 mM glyphosate). After two weeks growth on 1 mMglyphosate medium, cultures were transferred to the same base medium butwith 3 mM glyphosate. Additional transfers were made at approximately 2week intervals on 3 mM glyphosate medium. Glyphosate resistant calliwere identified approximately 8-10 weeks post-bombardment, at afrequency of approximately 0.2-1.0 resistant calli per bombarded plate.

Plants were regenerated from glyphosate resistant calli as described forbialaphos resistant calli in Example 10, except that instead of 1 mg/LBasta either 0.01 mM glyphosate or no selective agents were added to theculture medium. Plants were analyzed for expression of pMON19643 byWestern blot analysis. Leaf punches were taken from several individualplants regenerated from three independent glyphosate resistant calli.All three lines showed detectable levels of GOX gene expression. Fourplants assayed from line 264-2-1 had a low but detectable level of GOXexpression (approximately 0.002% of total protein). Five plants fromline 269-1-1 showed higher GOX protein levels ranging from 0.04-0.06% oftotal protein. Lastly, 23 plants were assayed from line 292-5-1. GOXprotein levels ranged from 0.05 to 0.1% of total protein. These plantssprayed with glyphosate at 29 oz./acre produced fully fertile plants. R₁progeny of these plants were sprayed with glyphosate at 29, 58 and 115oz/acre. One line of plants showed no vegetative damage at the highestapplication rate indicating glyphosate resistance at levels at whichcomplete weed control would be achieved.

Example 12

Effect of the HSP70 Intron Alterations

A. Deletions within the HSP70 intron

Deletion 1 (FIG. 2) (SEQ ID NO:2) was created by digestion of pMON19433with BsmI and NsiI, followed by treatment with T4 polymerase to createblunt ends, and religation. Deletion 2 (FIG. 3) (SEQ ID NO:3) was madesimilarly, except using digestion with BsmI and SnaBI. The effect ongene expression of the full length HSP70 intron versus the effect ofDeletion 1 or Deletion 2 was compared in BMS particle gun transientassays as described in Example 2. As shown below, the introns withinternal deletions increase GUS gene expression over the no introncontrol to a similar extent as the full length intron in pMON19433.

    ______________________________________    Intron            Relative GUS Expression    ______________________________________    none              1X    HSP70 full length 32-51X    HSP70 deletion 1  14-38X    HSP70 deletion 2  14-30X    ______________________________________

B. Alterations in 5' and 3' slice site consensus sequences

In the original polymerase chain reaction (PCR) synthesis of the HSP70intron by polymerase chain reaction, a variant intron was alsosynthesized. This variant intron, when cloned adjacent toβ-glucuronidase or luciferase, increases expression 4 fold relative to ano intron control but 10 fold less than the wild type HSP70 intron. Theonly significant difference in nucleotide sequence from that shown inSEQ ID NO:1 was a deletion of the adenine at nucleotide 19.

The HSP70 intron differs from the published (Brown, J. W. S., 1986, Nuc.Acid Res. 14:9949-9959) 5' splice site consensus sequence at twopositions and from the 3' splice site consensus sequence at oneposition. The deletion of nucleotide 19 causes the variant HSP70 intronto diverge from the 5' splice site consensus sequence at four positions.Thus, the variant intron probably does not splice as efficiently as thewildtype intron and this may account for the difference in their effecton gene expression.

To address this question, variants of the HSP70 intron that containperfect consensus sequences at the 5' splice junction, 3' splicejunction, or both were constructed. The variants of the HSP70 intronwere synthesized by PCR utilizing primers containing the desired changesto mutate the HSP70 intron splice sites to the 5' and/or 3' splicejunction consensus sequences. Specifically, the 5' splice junctionconsensus primer contained nucleotides 1 to 26 of SEQ ID NO:1 exceptthat nucleotide 15 and nucleotide 20 were each changed to adenine. The3' splice junction consensus primer contained nucleotides thatcomplement nucleotides 791 to 816, except that nucleotide 800 waschanged to a guanine (cytosine in the primer).

The PCR products containing the variant HSP70 introns were digested withBglII and NcoI and cloned into pMON8677, analogously to the constructionof pMON19433. Therefore, each vector contains, from 5' to 3', theenhanced CaMV35S promoter, HSP70 intron (original or variant),β-glucuronidase (GUS) coding sequence, and nopaline synthasepolyadenylation region. They are all identical except for the intron.pMON19433 contains the original HSP70 intron, pMON19460 contains the 5'splice site consensus variant intron, pMON19463 contains the 3' splicesite consensus variant intron, and pMON19464 contains a variant introncontaining both 5' and 3' splice site consensus sequences.

pMON19460, pMON19463, pMON19464, and pMON19433 were compared intransient gene expression assays in BMS cells as described in Example 2.As shown below, none of the variations in the HSP70 intron significantlyaltered GUS gene expression.

    ______________________________________           Splice junction    Vector   5'        3'        Relative GUS expression    ______________________________________    pMON19433             HSP70 wt  HSP70 wt  1X    PMON19460             consensus HSP70 wt  1.1-1.4X    pMON19463             HSP70 wt  consensus 1.1-1.4X    pMON19464             consensus consensus 1.6-1.7X    ______________________________________

C. Increasing the number of exon sequences does not effect the HSP70intron

The original HSP70 "intron" contains the entire intervening sequence aswell as 10 bases of exon 1 and 11 bases of exon 2. Because the intron isplaced in the 5' untranslated leader region between the enhanced CaMV35Spromoter and coding sequence, those 21 bases of exon sequence are leftbehind in the leader. PCR primers that give 50 nucleotides of the 3' endof HSP70 exon 1 and/or 28 nucleotides of the 5' end of HSP70 exon 2(Shah et al., 1985, In Cell and Mol. Biol. of Plant Stress. Alan R.Liss, Inc. pp.181-200) were used to synthesize introns containingdifferent amounts of exon sequences to determine if extra HSP70 exonsequences would affect the splicing efficiency and ability to increasegene expression.

The PCR products containing the various HSP70 introns with differentexon lengths were digested with BglII and NcoI and cloned into pMON8677,analogously to the construction of pMON19433. Therefore, each vectorcontains, from 5' to 3', the enhanced CaMV35S promoter, HSP70 intronplus surrounding exon sequences, β-glucuronidase (GUS) coding sequence,and nopaline synthase polyadenylation region. They are all identicalexcept for the length of the HSP70 exon surrounding the intron.

These vectors were then compared in transient gene expression assays inBMS cells as described in Example 2. As shown below, none of thevariations in the HSP70 intron significantly altered GUS geneexpression.

    ______________________________________    Vector  Exon 1     Exon 2  Relative GUS Expression    ______________________________________    19433   10 nt      11 nt   1X    19462   10 nt      28 nt   0.6-0.9X    19465   50 nt      11 nt   1.2-1.5X    19466   50 nt      28 nt   0.8-1.5X    ______________________________________

Example 13

HSP70 Intron Increases Gene Expression in Wheat Cells

To test the effect of introns on gene expression in wheat cells,transient gene expression assays were performed. C983 wheat suspensioncells (obtained from Dr. I. Vasil, Univ. of Florida) were plated andbombarded with β-glucuronidase vectors containing no intron (pMON8677),ADH1 intron (pMON8678), and the HSP70 intron (pMON19433) as describedfor corn suspension cells in Example 2. As shown below, the effect ofthe ADH1 and HSP70 introns on GUS expression in wheat cells iscomparable to that in corn cells. The ADH1 intron vector produces higherlevels of GUS expression expression than does the vector with no intron,but the HSP70 intron vector produces significantly higher levels ofexpression than the ADH1 intron vector.

    ______________________________________    Vector        Intron  Mean Relative GUS    ______________________________________    pMON8677      none    1X    pMON8678      ADH1    2X    pMON19433     HSP70   6-9X    ______________________________________

Example 14

The HSP70 Intron Increases Gene Expression in Rice

Rice tissue culture line 812M from rice strain 8706, an indica/japonicahybrid, was grown in MS medium. One day after subculture the cells weretransferred to Whatman filters for particle gun bombardment.Bombardments were performed with CaCl₂ /spermidine precipitated plasmidDNA using a PDS-1000 as described for BMS cells (Example 3). The cellswere allowed to express the introduced genes for two days and thenharvested. β-Glucuronidase (GUS) and luciferase (LUX) were assayed asdescribed, supra. As shown in Table 7, in duplicate experiments thepresence of the HSP70 intron in the 5' untranslated region increasesaverage GUS expression relative to LUX expression about 10 fold over theexpression observed with the vector without an intron.

                  TABLE 7    ______________________________________    Effect of HSP70 Intron in Rice    Vector          Intron  GUS/LUX    ______________________________________    pMON8677        none    15.5    pMON19433       HSP70   150.7    ______________________________________

Example 15

Expression of CP4 EPSPS Using HSP70 Intron Vectors

pMON19653 (FIG. 27) was constructed to test expression of the CP4 EPSPSgene (U.S. patent application Ser. No. 07/749,611 filed Aug. 28, 1991incorrporated herein by reference) in an HSP70 intron vector. A 1.7 kbBglII-EcoRI fragment containing the 300 bp chloroplast transit peptidefrom the Arabidopsis EPSPS gene (AEPSPS CTP) fused in frame to the 1.4kb bacterial CP4 EPSPS protein coding region was cloned into BamHI-EcoRIdigested pMON19470 to form pMON19653. Thus, pMON19653 is comprised of,from 5' to 3', the enhanced CaMV35S promoter, HSP70 intron, AEPSPSCTP/CP4 coding sequence, and nopaline synthase polyadenylation region ina pUC-like backbone containing an NPTII gene for kanamycin selection inbacteria.

pMON19653 was introduced into embryogenic cells in combination withpMON19643 and transformed calli selected on glyphosate medium asdescribed in Example 11. Glyphosate resistant embryogenic callus wereassayed by Western Blot analysis. The amount of CP4 protein expressedwas determined by comparison to standards of E. coli produced protein.Nine lines were generated. The CP4 expression levels ranged fromundetectable to 0.3% of the total protein in crude extracts made fromthe embryogenic callus, with an average value of 0.17%.

The above examples indicate that the use of vectors containing the HSP70intron would be expected to enhance the expression in monocot plants ofother DNA sequences encoding proteins.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 3    (2) INFORMATION FOR SEQ ID NO:1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 816 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: double    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (synthetic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    AGATCTACCGTCTTCGGTACGCGCTCACTCCGCCCTCTGCCTTTGTTACTGCCACGTTTC60    TCTGAATGCTCTCTTGTGTGGTGATTGCTGAGAGTGGTTTAGCTGGATCTAGAATTACAC120    TCTGAAATCGTGTTCTGCCTGTGCTGATTACTTGCCGTCCTTTGTAGCAGCAAAATATAG180    GGACATGGTAGTACGAAACGAAGATAGAACCTACACAGCAATACGAGAAATGTGTAATTT240    GGTGCTTAGCGGTATTTATTTAAGCACATGTTGGTGTTATAGGGCACTTGGATTCAGAAG300    TTTGCTGTTAATTTAGGCACAGGCTTCATACTACATGGGTCAATAGTATAGGGATTCATA360    TTATAGGCGATACTATAATAATTTGTTCGTCTGCAGAGCTTATTATTTGCCAAAATTAGA420    TATTCCTATTCTGTTTTTGTTTGTGTGCTGTTAAATTGTTAACGCCTGAAGGAATAAATA480    TAAATGACGAAATTTTGATGTTTATCTCTGCTCCTTTATTGTGACCATAAGTCAAGATCA540    GATGCACTTGTTTTAAATATTGTTGTCTGAAGAAATAAGTACTGACAGTATTTTGATGCA600    TTGATCTGCTTGTTTGTTGTAACAAAATTTAAAAATAAAGAGTTTCCTTTTTGTTGCTCT660    CCTTACCTCCTGATGGTATCTAGTATCTACCAACTGACACTATATTGCTTCTCTTTACAT720    ACGTATCTTGCTCGATGCCTTCTCCCTAGTGTTGACCAGTGTTACTCACATAGTCTTTGC780    TCATTTCATTGTAATGCAGATACCAAGCGGCCATGG816    (2) INFORMATION FOR SEQ ID NO:2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 283 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (synthetic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    AGATCTACCGTCTTCGGTACGCGCTCACTCCGCCCTCTGCCTTTGTTACTGCCACGTTTC60    TCTGAATGTGATCTGCTTGTTTGTTGTAACAAAATTTAAAAATAAAGAGTTTCCTTTTTG120    TTGCTCTCCTTACCTCCTGATGGTATCTAGTATCTACCAACTGACACTATATTGCTTCTC180    TTTACATACGTATCTTGCTCGATGCCTTCTCCCTAGTGTTGACCAGTGTTACTCACATAG240    TCTTTGCTCATTTCATTGTAATGCAGATACCAAGCGGCCATGG283    (2) INFORMATION FOR SEQ ID NO:3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 162 base pairs    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: DNA (synthetic)    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    AGATCTACCGTCTTCGGTACGCGCTCACTCCGCCCTCTGCCTTTGTTACTGCCACGTTTC60    TCTGAATGGTATCTTGCTCGATGCCTTCTCCCTAGTGTTGACCAGTGTTACTCACATAGT120    CTTTGCTCATTTCATTGTAATGCAGATACCAAGCGGCCATGG162    __________________________________________________________________________

We claim:
 1. An isolated DNA segment comprising in sequence:(a) apromotor that functions in monocotyledonous plant cells; (b) anon-translated leader DNA comprising an intron sequence including atleast sufficient nucleotide sequence from the 5' end and from the 3' endof SEQ ID NO: 1 to splice said intron sequence; and (c) a DNA sequencethat in combination with (a) and (b) effects production of an RNAsequence; wherein the non-translated leader of (b) increases expressionof the DNA sequence relative to wild-type expression.
 2. The isolatedDNA segment of claim 1, further comprising a 3' non-translated sequencethat functions in plant cells to cause addition of a polyadenylatednucleotide sequence to the 3' end of said RNA sequence.
 3. The isolatedDNA segment of claim 1, wherein said intron sequence comprises at leastabout 162 nucleotides created by ligating a 5' end sequence and a 3' endsequence from SEQ ID NO:1.
 4. The isolated DNA segment of claim 3,wherein said intron sequence is SEQ ID NO:3.
 5. The isolated DNA segmentof claim 3, wherein said intron sequence comprises at least about 283nucleotides created by ligating a 5' end sequence and a 3' end sequencefrom SEQ ID NO:
 1. 6. The isolated DNA segment of claim 5, wherein saidintron sequence is SEQ ID NO:2.
 7. The isolated DNA segment of claim 5,wherein said intron sequence is SEQ ID NO:
 1. 8. The isolated DNAsegment of claim 1, fused to said leader DNA further comprising betweenabout 10 and about 50 additional nucleotides of an exon fused to saidleader DNA.
 9. The isolated DNA segment of claim 1, further comprisingbetween about 11 and about 28 additional nucleotides of an exon fused tosaid leader DNA.
 10. The isolated DNA segment of claim 1, furthercomprising between about 10 and about 50 additional nucleotides from the3' end of HSP70 exon 1 and between about 11 and about 28 additionalnucleotides from the 5' end of HSP70 exon 2 fused to said leader DNA.11. The isolated DNA segment of claim 1, wherein said intron sequencecomprises a splice site consensus sequence.
 12. The isolated DNA segmentof claim 11, wherein said intron sequence comprises a 5' splice siteconsensus sequence.
 13. The isolated DNA segment of claim 11, whereinsaid intron sequence comprises a 3' splice site consensus sequence. 14.The isolated DNA segment of claim 11, wherein said intron sequencecomprises a 5' splice site consensus sequence and a 3' splice siteconsensus sequence.
 15. The isolated DNA segment of claim 11, whereinsaid intron sequence has an adenine nucleotide at position 15 of SEQ IDNO:
 1. 16. The isolated DNA segment of claim 11, wherein said intronsequence has an adenine nucleotide at position 20 of SEQ ID NO:
 1. 17.The isolated DNA segment of claim 11, wherein said intron sequence has aguanine nucleotide at position 800 of SEQ ID NO:
 1. 18. The isolated DNAsegment of claim 14, wherein said intron sequence has an adeninenucleotide at position 15, an adenine nucleotide at position 20, and aguanine nucleotide at position 800 of SEQ ID NO:
 1. 19. The isolated DNAsegment of claim 1, wherein said promoter comprises a plant DNA viruspromoter.
 20. The isolated DNA segment of claim 14, wherein saidpromoter is a CaMV35S promoter or an FMV promoter.
 21. The isolated DNAsegment of claim 1, wherein said DNA sequence encodes an EPSP synthase,a CP4 protein, an ACC-deaminase, a B.t. crystal toxin, a glgC16 protein,a plant viral coat protein or a GOX protein.
 22. A method for theexpression of a gene in a monocotyledonous plant, comprising introducinginto the plant cell a DNA segment comprising in sequence:(a) a promotorthat functions in monocotyledonous plant cells; (b) a non-translatedleader comprising an intron sequence including at least sufficientnucleotide sequence from the 5' end and from the 3' end of SEQ ID NO: 1to splice said intron; and (c) a DNA sequence comprising said gene;wherein the DNA segment comprising (a), (b) and (c) produces an RNAsequence that effects expression of said gene.
 23. The method of claim22, wherein said DNA further comprises a 3' non-translated sequence thatfunctions in monocotyledonous plant cells to cause addition of apolyadenylated nucleotide sequence to the 3' end of said RNA sequence.24. The method of claim 22, wherein said intron sequence comprises atleast about 162 nucleotides comprised by ligating a 5' end sequence anda 3' end sequence from SEQ ID NO:
 1. 25. The method of claim 22, furthercomprising between about 10 and about 50 additional nucleotides of anexon fused to said leader DNA.
 26. The method of claim 22, wherein saidintron sequence comprises a splice site consensus sequence.
 27. Themethod of claim 22, wherein said DNA sequence encodes an EPSP synthase,a CP4 protein, an ACC-deaminase, a B.t. crystal toxin, a glgC16 protein,a plant viral coat protein or a GOX protein.
 28. The method of claim 22,wherein said plant is maize, wheat or rice.
 29. A transgenicmonocotyledonous plant comprising the DNA of claim
 11. 30. The plant ofclaim 29, wherein said DNA further comprises a 3' non-translatedsequence that functions in plant cells to cause addition of apolyadenylated nucleotide sequence to the 3' end of said RNA sequence.31. The plant of claim 29, wherein said intron sequence comprises atleast about 162 nucleotides obtained by ligating a 5° end sequence and a3° end sequence from SEQ ID NO:
 1. 32. The plant of claim 29, furthercomprising between about 10 and about 50 additional nucleotides of anexon fused to said leader DNA.
 33. The plant of claim 29, wherein saidintron sequence comprises a splice site consensus sequence.
 34. Theplant of claim 29, wherein said DNA sequence encodes an EPSP synthase, aCP4 protein, an ACC-deaminase, a B.t. crystal toxin, a glgC16 protein, aplant viral coat protein or a GOX protein.
 35. The plant of claim 29,wherein said plant is maize, wheat or rice.